Compositions and methods for siRNA inhibition of angiogenesis

ABSTRACT

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/042,040, filed Sep. 30, 2013, now U.S. Pat. No. 8,946,403, which is acontinuation of U.S. application Ser. No. 11/422,982, filed on Jun. 8,2006, now U.S. Pat. No. 8,546,345, which is a divisional of U.S.application Ser. No. 10/294,228, filed on Nov. 14, 2002, now U.S. Pat.No. 7,148,342, which claims the benefit of U.S. Provisional ApplicationSer. No. 60/398,417, filed on Jul. 24, 2002, each of which are herebyincorporated herein by reference in their entireties.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported in part by NIH/NEI GrantNo. R01-EY10820, EY-13410 and EY12156. The U.S. government has certainrights in this invention.

JOINT RESEARCH AGREEMENT

Not Applicable

INCORPORATION OF SEQUENCE LISTING

A computer readable form of the sequence listing submitted in U.S.patent application Ser. No. 10/294,228, which was created on Nov. 14,2002, is herein incorporated by reference. The information in theelectronic format of the sequence listing is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to the regulation of gene expression by smallinterfering RNA, in particular for treating diseases or conditionsinvolving angiogenesis.

BACKGROUND OF THE INVENTION

Angiogenesis, defined as the growth of new capillary blood vessels or“neovascularization,” plays a fundamental role in growth anddevelopment. In mature humans, the ability to initiate angiogenesis ispresent in all tissues, but is held under strict control. A keyregulator of angiogenesis is vascular endothelial growth factor(“VEGF”), also called vascular permeability factor (“VPF”). VEGF existsin at least four different alternative splice forms in humans (VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆), all of which exert similar biologicalactivities.

Angiogenesis is initiated when secreted VEGF binds to the Flt-1 andFlk-1/KDR receptors (also called VEGF receptor 1 and VEGF receptor 2),which are expressed on the surface of endothelial cells. Flt-1 andFlk-1/KDR are transmembrane protein tyrosine kinases, and binding ofVEGF initiates a cell signal cascade resulting in the ultimateneovascularization in the surrounding tissue.

Aberrant angiogenesis, or the pathogenic growth of new blood vessels, isimplicated in a number of conditions. Among these conditions arediabetic retinopathy, psoriasis, exudative or “wet” age-related maculardegeneration (“ARMD”), rheumatoid arthritis and other inflammatorydiseases, and most cancers. The diseased tissues or tumors associatedwith these conditions express abnormally high levels of VEGF, and show ahigh degree of vascularization or vascular permeability.

ARMD in particular is a clinically important angiogenic disease. Thiscondition is characterized by choroidal neovascularization in one orboth eyes in aging individuals, and is the major cause of blindness inindustrialized countries.

A number of therapeutic strategies exist for inhibiting aberrantangiogenesis, which attempt to reduce the production or effect of VEGF.For example, anti-VEGF or anti-VEGF receptor antibodies (Kim E S et al.(2002), PNAS USA 99: 11399-11404), and soluble VEGF “traps” whichcompete with endothelial cell receptors for VEGF binding (Holash J etal. (2002), PNAS USA 99: 11393-11398) have been developed. ClassicalVEGF “antisense” or aptamer therapies directed against VEGF geneexpression have also been proposed (U.S. published application2001/0021772 of Uhlmann et al.). However, the anti-angiogenic agentsused in these therapies can produce only a stoichiometric reduction inVEGF or VEGF receptor, and the agents are typically overwhelmed by theabnormally high production of VEGF by the diseased tissue. The resultsachieved with available anti-angiogenic therapies have therefore beenunsatisfactory.

RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e., <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell(Fire A et al. (1998), Nature 391: 806-811). These short dsRNAmolecules, called “short interfering RNA” or “siRNA,” cause thedestruction of messenger RNAs (“mRNAs”) which share sequence homologywith the siRNA to within one nucleotide resolution (Elbashir S M et al.(2001), Genes Dev, 15: 188-200). It is believed that the siRNA and thetargeted mRNA bind to an “RNA-induced silencing complex” or “RISC”,which cleaves the targeted mRNA. The siRNA is apparently recycled muchlike a multiple-turnover enzyme, with 1 siRNA molecule capable ofinducing cleavage of approximately 1000 mRNA molecules. siRNA-mediatedRNAi degradation of an mRNA is therefore more effective than currentlyavailable technologies for inhibiting expression of a target gene.

Elbashir S M et al. (2001), supra, has shown that synthetic siRNA of 21and 22 nucleotides in length, and which have short 3′ overhangs, areable to induce RNAi of target mRNA in a Drosophila cell lysate. Culturedmammalian cells also exhibit RNAi degradation with synthetic siRNA(Elbashir S M et al. (2001) Nature, 411: 494-498), and RNAi degradationinduced by synthetic siRNA has recently been shown in living mice(McCaffrey A P et al. (2002), Nature, 418: 38-39; Xia H et al. (2002),Nat. Biotech. 20: 1006-1010). The therapeutic potential of siRNA-inducedRNAi degradation has been demonstrated in several recent in vitrostudies, including the siRNA-directed inhibition of HIV-1 infection(Novina C D et al. (2002), Nat. Med. 8: 681-686) and reduction ofneurotoxic polyglutamine disease protein expression (Xia H et al.(2002), supra).

What is needed, therefore, are agents which selectively inhibitexpression of VEGF or VEGF receptors in catalytic or sub-stoichiometricamounts.

SUMMARY OF THE INVENTION

The present invention is directed to siRNAs which specifically targetand cause RNAi-induced degradation of mRNA from VEGF, Flt-1 andFlk-1/KDR genes. The siRNA compounds and compositions of the inventionare used to inhibit angiogenesis, in particular for the treatment ofcancerous tumors, age-related macular degeneration, and other angiogenicdiseases.

Thus, the invention provides an isolated siRNA which targets human VEGFmRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or an alternative spliceform, mutant or cognate thereof. The siRNA comprises a sense RNA strandand an antisense RNA strand which form an RNA duplex. The sense RNAstrand comprises a nucleotide sequence identical to a target sequence ofabout 19 to about 25 contiguous nucleotides in the target mRNA.

The invention also provides recombinant plasmids and viral vectors whichexpress the siRNA of the invention, as well as pharmaceuticalcompositions comprising the siRNA of the invention and apharmaceutically acceptable carrier.

The invention further provides a method of inhibiting expression ofhuman VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or analternative splice form, mutant or cognate thereof, comprisingadministering to a subject an effective amount of the siRNA of theinvention such that the target mRNA is degraded.

The invention further provides a method of inhibiting angiogenesis in asubject, comprising administering to a subject an effective amount of ansiRNA targeted to human VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof.

The invention further provides a method of treating an angiogenicdisease, comprising administering to a subject in need of such treatmentan effective amount of an siRNA targeted to human VEGF mRNA, human Flt-1mRNA, human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof, such that angiogenesis associated with the angiogenicdisease is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a histograms of VEGF concentration (in pg/ml) inhypoxic 293 and HeLa cells treated with no siRNA (“-”); nonspecificsiRNA (“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). VEGFconcentration (in pg/ml) in non-hypoxic 293 and HeLa cells is alsoshown. Each bar represents the average of four experiments, and theerror is the standard deviation of the mean.

FIG. 2 is a histogram of murine VEGF concentration (in pg/ml) in hypoxicNIH 3T3 cells treated with no siRNA (“-”); nonspecific siRNA(“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). Each barrepresents the average of six experiments and the error is the standarddeviation of the mean.

FIG. 3 is a histogram of human VEGF concentration (pg/total protein) inretinas from mice injected with adenovirus expressing human VEGF(“AdVEGF”) in the presence of either GFP siRNA (dark gray bar) or humanVEGF siRNA (light grey bar). Each bar represent the average of 5 eyesand the error bars represent the standard error of the mean.

FIG. 4 is a histogram showing the mean area (in mm²) of laser-inducedCNV in control eyes given subretinal injections of GFP siRNA (N=9; “GFPsiRNA”), and in eyes given subretinal injections of mouse VEGF siRNA(N=7; “Mouse VEGF siRNA”). The error bars represent the standard errorof the mean.

FIG. 5 is a schematic representation of pAAVsiRNA, a cis-acting plasmidused to generate a recombinant AAV viral vector of the invention. “ITR”:AAV inverted terminal repeats; “U6”: U6 RNA promoters; “Sense”: siRNAsense coding sequence; “Anti”: siRNA antisense coding sequence; “PolyT”:polythymidine termination signals.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all nucleic acid sequences herein are givenin the 5′ to 3′ direction. Also, all deoxyribonucleotides in a nucleicacid sequence are represented by capital letters (e.g., deoxythymidineis “T”), and ribonucleotides in a nucleic acid sequence are representedby lower case letters (e.g., uridine is “u”).

Compositions and methods comprising siRNA targeted to VEGF, Flt-1 orFlk-1/KDR mRNA are advantageously used to inhibit angiogenesis, inparticular for the treatment of angiogenic disease. The siRNA of theinvention are believed to cause the RNAi-mediated degradation of thesemRNAs, so that the protein product of the VEGF, Flt-1 or Flk-1/KDR genesis not produced or is produced in reduced amounts. Because VEGF bindingto the Flt-1 or Flk-1/KDR receptors is required for initiating andmaintaining angiogenesis, the siRNA-mediated degradation of VEGF, Flt-1or Flk-1/KDR mRNA inhibits the angiogenic process.

The invention therefore provides isolated siRNA comprising shortdouble-stranded RNA from about 17 nucleotides to about 29 nucleotides inlength, preferably from about 19 to about 25 nucleotides in length, thatare targeted to the target mRNA. The siRNA comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Asis described in more detail below, the sense strand comprises a nucleicacid sequence which is identical to a target sequence contained withinthe target mRNA.

The sense and antisense strands of the present siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. Without wishingto be bound by any theory, it is believed that the hairpin area of thelatter type of siRNA molecule is cleaved intracellularly by the “Dicer”protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra).

As used herein, “isolated” means altered or removed from the naturalstate through human intervention. For example, an siRNA naturallypresent in a living animal is not “isolated,” but a synthetic siRNA, oran siRNA partially or completely separated from the coexisting materialsof its natural state is “isolated.” An isolated siRNA can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a cell into which the siRNA has been delivered.

As used herein, “target mRNA” means human VEGF, Flt-1 or Flk-1/KDR mRNA,mutant or alternative splice forms of human VEGF, Flt-1 or Flk-1/KDRmRNA, or mRNA from cognate VEGF, Flt-1 or Flk-1/KDR genes.

As used herein, a gene or mRNA which is “cognate” to human VEGF, Flt-1or Flk-1/KDR is a gene or mRNA from another mammalian species which ishomologous to human VEGF, Flt-1 or Flk-1/KDR. For example, the cognateVEGF mRNA from the mouse is given in SEQ ID NO: 1.

Splice variants of human VEGF are known, including VEGF₁₂₁ (SEQ ID NO:2), VEGF₁₆₅ (SEQ ID NO: 3), VEGF₁₈₉ (SEQ ID NO: 4) and VEGF₂₀₆ (SEQ IDNO: 5). The mRNA transcribed from the human VEGF, Flt-1 (SEQ ID NO: 6)or Flk-1/KDR (SEQ ID NO: 7) genes can be analyzed for furtheralternative splice forms using techniques well-known in the art. Suchtechniques include reverse transcription-polymerase chain reaction(RT-PCR), northern blotting and in-situ hybridization. Techniques foranalyzing mRNA sequences are described, for example, in Busting S A(2000), J. Mol. Endocrinol. 25: 169-193, the entire disclosure of whichis herein incorporated by reference. Representative techniques foridentifying alternatively spliced mRNAs are also described below.

For example, databases that contain nucleotide sequences related to agiven disease gene can be used to identify alternatively spliced mRNA.Such databases include GenBank, Embase, and the Cancer Genome AnatomyProject (CGAP) database. The CGAP database, for example, containsexpressed sequence tags (ESTs) from various types of human cancers. AnmRNA or gene sequence from the VEGF, Flt-1 or Flk-1/KDR genes can beused to query such a database to determine whether ESTs representingalternatively spliced mRNAs have been found for a these genes.

A technique called “RNAse protection” can also be used to identifyalternatively spliced VEGF, Flt-1 or Flk-1/KDR mRNAs. RNAse protectioninvolves translation of a gene sequence into synthetic RNA, which ishybridized to RNA derived from other cells; for example, cells fromtissue at or near the site of neovascularization. The hybridized RNA isthen incubated with enzymes that recognize RNA:RNA hybrid mismatches.Smaller than expected fragments indicate the presence of alternativelyspliced mRNAs. The putative alternatively spliced mRNAs can be clonedand sequenced by methods well known to those skilled in the art.

RT-PCR can also be used to identify alternatively spliced VEGF, Flt-1 orFlk-1/KDR mRNAs. In RT-PCR, mRNA from the diseased tissue is convertedinto cDNA by the enzyme reverse transcriptase, using methods well-knownto those of ordinary skill in the art. The entire coding sequence of thecDNA is then amplified via PCR using a forward primer located in the 3′untranslated region, and a reverse primer located in the 5′ untranslatedregion. The amplified products can be analyzed for alternative spliceforms, for example by comparing the size of the amplified products withthe size of the expected product from normally spliced mRNA, e.g., byagarose gel electrophoresis. Any change in the size of the amplifiedproduct can indicate alternative splicing.

mRNA produced from mutant VEGF, Flt-1 or Flk-1/KDR genes can also bereadily identified through the techniques described above foridentifying alternative splice forms. As used herein, “mutant” VEGF,Flt-1 or Flk-1/KDR genes or mRNA include human VEGF, Flt-1 or Flk-1/KDRgenes or mRNA which differ in sequence from the VEGF, Flt-1 or Flk-1/KDRsequences set forth herein. Thus, allelic forms of these genes, and themRNA produced from them, are considered “mutants” for purposes of thisinvention.

It is understood that human VEGF, Flt-1 or Flk-1/KDR mRNA may containtarget sequences in common with their respective alternative spliceforms, cognates or mutants. A single siRNA comprising such a commontargeting sequence can therefore induce RNAi-mediated degradation ofdifferent RNA types which contain the common targeting sequence.

The siRNA of the invention can comprise partially purified RNA,substantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, including modifications that make the siRNAresistant to nuclease digestion.

One or both strands of the siRNA of the invention can also comprise a 3′overhang. As used herein, a “3′ overhang” refers to at least oneunpaired nucleotide extending from the 3′-end of a duplexed RNA strand.

Thus in one embodiment, the siRNA of the invention comprises at leastone 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxynucleotides) in length, preferably from 1 toabout 5 nucleotides in length, more preferably from 1 to about 4nucleotides in length, and particularly preferably from about 2 to about4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the invention can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

In certain embodiments, the siRNA of the invention comprises thesequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNAcomprise approximately 30-70% GC, and preferably comprise approximately50% G/C. The sequence of the sense siRNA strand corresponds to (N19)TTor N21 (i.e., positions 3 to 23), respectively. In the latter case, the3′ end of the sense siRNA is converted to TT. The rationale for thissequence conversion is to generate a symmetric duplex with respect tothe sequence composition of the sense and antisense strand 3′ overhangs.The antisense RNA strand is then synthesized as the complement topositions 1 to 21 of the sense strand.

Because position 1 of the 23-nt sense strand in these embodiments is notrecognized in a sequence-specific manner by the antisense strand, the3′-most nucleotide residue of the antisense strand can be chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

In another embodiment, the siRNA of the invention comprises the sequenceNAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine(e.g., C or U/T). The respective 21-nt sense and antisense RNA strandsof this embodiment therefore generally begin with a purine nucleotide.Such siRNA can be expressed from pol III expression vectors without achange in targeting site, as expression of RNAs from pol III promotersis only believed to be efficient when the first transcribed nucleotideis a purine.

The siRNA of the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T et al., “ThesiRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Department of Cellular Biochemistry, AG 105, Max-Planck-Institute forBiophysical Chemistry, 37077 Göttingen, Germany, and can be found byaccessing the website of the Max Planck Institute and searching with thekeyword “siRNA.” Thus, the sense strand of the present siRNA comprises anucleotide sequence identical to any contiguous stretch of about 19 toabout 25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon (see,e.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below,which are within 100 nt of the 5′-end of the VEGF₁₂₁ cDNA

For example, a suitable target sequence in the VEGF₁₂₁ cDNA sequence is:

(SEQ ID NO: 8) TCATCACGAAGTGGTGAAG

Thus, an siRNA of the invention targeting this sequence, and which has3′ uu overhangs on each strand (overhangs shown in bold), is:

(SEQ ID NO: 9) 5′-ucaucacgaaguggugaaguu-3′ (SEQ ID NO: 10)3′-uuaguagugcuucaccacuuc-5′

An siRNA of the invention targeting this same sequence, but having 3′ TToverhangs on each strand (overhangs shown in bold) is:

(SEQ ID NO: 11) 5′-ucaucacgaaguggugaagTT-3′ (SEQ ID NO: 12)3′-TTaguagugcuucaccacuuc-5′

Other VEGF₁₂₁ target sequences from which siRNA of the invention can bederived are given in Table 1. It is understood that all VEGF₁₂₁ targetsequences listed in Table 1 are within that portion of the VEGF₁₂₁alternative splice form which is common to all human VEGF alternativesplice forms. Thus, the VEGF₁₂₁ target sequences in Table 1 can alsotarget VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆ mRNA. Target sequences which targeta specific VEGF isoform can also be readily identified. For example, atarget sequence which targets VEGF₁₆₅ mRNA but not VEGF₁₂₁ mRNA isAACGTACTTGCAGATGTGACA (SEQ ID NO: 13).

TABLE 1 VEGF Target Sequences SEQ target sequence ID NO:GTTCATGGATGTCTATCAG 14 TCGAGACCCTGGTGGACAT 15 TGACGAGGGCCTGGAGTGT 16TGACGAGGGCCTGGAGTGT 17 CATCACCATGCAGATTATG 18 ACCTCACCAAGGCCAGCAC 19GGCCAGCACATAGGAGAGA 20 CAAATGTGAATGCAGACCA 21 ATGTGAATGCAGACCAAAG 22TGCAGACCAAAGAAAGATA 23 AGAAAGATAGAGCAAGACA 24 GAAAGATAGAGCAAGACAA 25GATAGAGCAAGACAAGAAA 26 GACAAGAAAATCCCTGTGG 27 GAAAATCCCTGTGGGCCTT 28AATCCCTGTGGGCCTTGCT 29 TCCCTGTGGGCCTTGCTCA 30 GCATTTGTTTGTACAAGAT 31GATCCGCAGACGTGTAAAT 32 ATGTTCCTGCAAAAACACA 33 TGTTCCTGCAAAAACACAG 34AAACACAGACTCGCGTTGC 35 AACACAGACTCGCGTTGCA 36 ACACAGACTCGCGTTGCAA 37CACAGACTCGCGTTGCAAG 38 GGCGAGGCAGCTTGAGTTA 39 ACGAACGTACTTGCAGATG 40CGAACGTACTTGCAGATGT 41 CGTACTTGCAGATGTGACA 42 GTGGTCCCAGGCTGCACCC 43GGAGGAGGGCAGAATCATC 44 GTGGTGAAGTTCATGGATG 45 AATCATCACGAAGTGGTGAAG 46AAGTTCATGGATGTCTATCAG 47 AATCGAGACCCTGGTGGACAT 48 AATGACGAGGGCCTGGAGTGT49 AACATCACCATGCAGATTATG 50 AAACCTCACCAAGGCCAGCAC 51AAGGCCAGCACATAGGAGAGA 52 AACAAATGTGAATGCAGACCA 53 AAATGTGAATGCAGACCAAAG54 AATGCAGACCAAAGAAAGATA 55 AAAGAAAGATAGAGCAAGACA 56AAGAAAGATAGAGCAAGACAA 57 AAGATAGAGCAAGACAAGAAAAT 58AAGACAAGAAAATCCCTGTGGGC 59 AAGAAAATCCCTGTGGGCCTTGC 60AATCCCTGTGGGCCTTGCTCAGA 61 AAGCATTTGTTTGTACAAGATCC 62AAGATCCGCAGACGTGTAAATGT 63 AAATGTTCCTGCAAAAACACAGA 64AATGTTCCTGCAAAAACACAGAC 65 AAAAACACAGACTCGCGTTGCAA 66AAAACACAGACTCGCGTTGCAAG 67 AAACACAGACTCGCGTTGCAAGG 68AACACAGACTCGCGTTGCAAGGC 69 AAGGCGAGGCAGCTTGAGTTAAA 70AAACGAACGTACTTGCAGATGTG 71 AACGAACGTACTTGCAGATGTGA 72AAGTGGTCCCAGGCTGCACCCAT 73 AAGGAGGAGGGCAGAATCATCAC 74AAGTGGTGAAGTTCATGGATGTC 75 AAAATCCCTGTGGGCCTTGCTCA 76GGCAGAATCATCACGAAGTGG 81 CCTGGTGGACATCTTCCAGGA 82 GAGATCGAGTACATCTTCAAG83 TGGAGTGTGTGCCCACTGAGG 84 GAGCTTCCTACAGCACAACAA 85TTGCTCAGAGCGGAGAAAGCA 86 CACACACTCGCGTTGCAAGGC 87 TCACCATGCAGATTATGCGGA88 TAGAGCAAGACAAGAAAATCC 89 CCGCAGACGTGTAAATGTTCC 90

The siRNA of the invention can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA can bechemically synthesized or recombinantly produced using methods known inthe art, such as the Drosophila in vitro system described in U.S.published application 2002/0086356 of Tuschl et al., the entiredisclosure of which is herein incorporated by reference.

Preferably, the siRNA of the invention are chemically synthesized usingappropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va.,USA), ChemGenes (Ashland, Mass., USA) and Cruachem (Glasgow, UK).

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Suitable promoters forexpressing siRNA of the invention from a plasmid include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant plasmids of the invention can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly at or near the area of neovascularization invivo. The use of recombinant plasmids to deliver siRNA of the inventionto cells in vivo is discussed in more detail below.

siRNA of the invention can be expressed from a recombinant plasmideither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Selection of plasmids suitable for expressing siRNA of the invention,methods for inserting nucleic acid sequences for expressing the siRNAinto the plasmid, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for exampleTuschl, T. (2002), Nat. Biotechnol, 20: 446-448; Brummelkamp T R et al.(2002), Science 296: 550-553; Miyagishi M et al. (2002), Nat.Biotechnol. 20: 497-500; Paddison P J et al. (2002), Genes Dev. 16:948-958; Lee N S et al. (2002), Nat. Biotechnol. 20: 500-505; and Paul CP et al. (2002), Nat. Biotechnol. 20: 505-508, the entire disclosures ofwhich are herein incorporated by reference.

A plasmid comprising nucleic acid sequences for expressing an siRNA ofthe invention is described in Example 7 below. That plasmid, calledpAAVsiRNA, comprises a sense RNA strand coding sequence in operableconnection with a polyT termination sequence under the control of ahuman U6 RNA promoter, and an antisense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. The plasmid pAAVsiRNA is ultimately intendedfor use in producing an recombinant adeno-associated viral vectorcomprising the same nucleic acid sequences for expressing an siRNA ofthe invention.

As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

As used herein, “under the control” of a promoter means that the nucleicacid sequences encoding the sense or antisense strands are located 3′ ofthe promoter, so that the promoter can initiate transcription of thesense or antisense coding sequences.

The siRNA of the invention can also be expressed from recombinant viralvectors intracellularly at or near the area of neovascularization invivo. The recombinant viral vectors of the invention comprise sequencesencoding the siRNA of the invention and any suitable promoter forexpressing the siRNA sequences. Suitable promoters include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the siRNAin a particular tissue or in a particular intracellular environment. Theuse of recombinant viral vectors to deliver siRNA of the invention tocells in vivo is discussed in more detail below.

siRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of the viral vectors can also be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses. For example, an AAV vector of the inventioncan be pseudotyped with surface proteins from vesicular stomatitis virus(VSV), rabies, Ebola, Mokola, and the like.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the siRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the siRNA of the invention, methodsfor constructing the recombinant AAV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol., 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference. Anexemplary method for generating a recombinant AAV vector of theinvention is described in Example 7 below.

The ability of an siRNA containing a given target sequence to causeRNAi-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, siRNA of the invention can be delivered to cultured cells,and the levels of target mRNA can be measured by Northern blot or dotblotting techniques, or by quantitative RT-PCR. Alternatively, thelevels of VEGF, Flt-1 or Flk-1/KDR receptor protein in the culturedcells can be measured by ELISA or Western blot. A suitable cell culturesystem for measuring the effect of the present siRNA on target mRNA orprotein levels is described in Example 1 below.

RNAi-mediated degradation of target mRNA by an siRNA containing a giventarget sequence can also be evaluated with animal models ofneovascularization, such as the ROP or CNV mouse models. For example,areas of neovascularization in an ROP or CNV mouse can be measuredbefore and after administration of an siRNA. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates the down-regulation of the target mRNA (see Example 6 below).

As discussed above, the siRNA of the invention target and cause theRNAi-mediated degradation of VEGF, Flt-1 or Flk-1/KDR mRNA, oralternative splice forms, mutants or cognates thereof. Degradation ofthe target mRNA by the present siRNA reduces the production of afunctional gene product from the VEGF, Flt-1 or Flk-1/KDR genes. Thus,the invention provides a method of inhibiting expression of VEGF, Flt-1or Flk-1/KDR in a subject, comprising administering an effective amountof an siRNA of the invention to the subject, such that the target mRNAis degraded. As the products of the VEGF, Flt-1 and Flk-1/KDR genes arerequired for initiating and maintaining angiogenesis, the invention alsoprovides a method of inhibiting angiogenesis in a subject by theRNAi-mediated degradation of the target mRNA by the present siRNA.

As used herein, a “subject” includes a human being or non-human animal.Preferably, the subject is a human being.

As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit the progression of angiogenesis in asubject.

RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

Inhibition of angiogenesis can be evaluated by directly measuring theprogress of pathogenic or nonpathogenic angiogenesis in a subject; forexample, by observing the size of a neovascularized area before andafter treatment with the siRNA of the invention. An inhibition ofangiogenesis is indicated if the size of the neovascularized area staysthe same or is reduced. Techniques for observing and measuring the sizeof neovascularized areas in a subject are within the skill in the art;for example, areas of choroid neovascularization can be observed byophthalmoscopy.

Inhibition of angiogenesis can also be inferred through observing achange or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA of the invention.

It is understood that the siRNA of the invention can degrade the targetmRNA (and thus inhibit angiogenesis) in substoichiometric amounts.Without wishing to be bound by any theory, it is believed that the siRNAof the invention causes degradation of the target mRNA in a catalyticmanner. Thus, compared to standard anti-angiogenic therapies,significantly less siRNA needs to be delivered at or near the site ofneovascularization to have a therapeutic effect.

One skilled in the art can readily determine an effective amount of thesiRNA of the invention to be administered to a given subject, by takinginto account factors such as the size and weight of the subject; theextent of the neovascularization or disease penetration; the age, healthand sex of the subject; the route of administration; and whether theadministration is regional or systemic. Generally, an effective amountof the siRNA of the invention comprises an intercellular concentrationat or near the neovascularization site of from about 1 nanomolar (nM) toabout 100 nM, preferably from about 2 nM to about 50 nM, more preferablyfrom about 2.5 nM to about 10 nM. It is contemplated that greater orlesser amounts of siRNA can be administered.

The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendometrial neovascularization, and processes involved in the productionof fatty tissues or cholesterol. Thus, the invention provides a methodfor inhibiting non-pathogenic angiogenesis, e.g., for controlling weightor promoting fat loss, for reducing cholesterol levels, or as anabortifacient.

The present methods can also inhibit angiogenesis which is associatedwith an angiogenic disease; i.e., a disease in which pathogenicity isassociated with inappropriate or uncontrolled angiogenesis. For example,most cancerous solid tumors generate an adequate blood supply forthemselves by inducing angiogenesis in and around the tumor site. Thistumor-induced angiogenesis is often required for tumor growth, and alsoallows metastatic cells to enter the bloodstream.

Other angiogenic diseases include diabetic retinopathy, age-relatedmacular degeneration (ARMD), psoriasis, rheumatoid arthritis and otherinflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

Preferably, an siRNA of the invention is used to inhibit the growth ormetastasis of solid tumors associated with cancers; for example breastcancer, lung cancer, head and neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer(e.g., melanoma), lymphomas and blood cancer.

More preferably, an siRNA of the invention is used to inhibit choroidalneovascularization in age-related macular degeneration.

For treating angiogenic diseases, the siRNA of the invention canadministered to a subject in combination with a pharmaceutical agentwhich is different from the present siRNA. Alternatively, the siRNA ofthe invention can be administered to a subject in combination withanother therapeutic method designed to treat the angiogenic disease. Forexample, the siRNA of the invention can be administered in combinationwith therapeutic methods currently employed for treating cancer orpreventing tumor metastasis (e.g., radiation therapy, chemotherapy, andsurgery). For treating tumors, the siRNA of the invention is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplatin, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the siRNA to a particular tissue,such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

Preferably, the liposomes encapsulating the present siRNA comprises aligand molecule that can target the liposome to a particular cell ortissue at or near the site of angiogenesis. Ligands which bind toreceptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present siRNAare modified so as to avoid clearance by the mononuclear macrophage andreticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment, a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Liposomes modified with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or“leaky” microvasculature. Thus, target tissue characterized by suchmicrovasculature defects, for example solid tumors, will efficientlyaccumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA,18: 6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen. Thus, liposomes of the invention that are modifiedwith opsonization-inhibition moieties can deliver the present siRNA totumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate; synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g., polyvinylalcoholand polyxylitol to which carboxylic or amino groups are chemicallylinked, as well as gangliosides, such as ganglioside GM₁. Copolymers ofPEG, methoxy PEG, or methoxy PPG, or derivatives thereof, are alsosuitable. In addition, the opsonization inhibiting polymer can be ablock copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginicacid, carrageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Recombinant plasmids which express siRNA of the invention are discussedabove. Such recombinant plasmids can also be administered directly or inconjunction with a suitable delivery reagent, including the MirusTransit LT1 lipophilic reagent; lipofectin; lipofectamine; cellfectin;polycations (e.g., polylysine) or liposomes. Recombinant viral vectorswhich express siRNA of the invention are also discussed above, andmethods for delivering such vectors to an area of neovascularization ina patient are within the skill in the art.

The siRNA of the invention can be administered to the subject by anymeans suitable for delivering the siRNA to the cells of the tissue at ornear the area of neovascularization. For example, the siRNA can beadministered by gene gun, electroporation, or by other suitableparenteral or enteral administration routes.

Suitable enteral administration routes include oral, rectal, orintranasal delivery.

Suitable parenteral administration routes include intravascularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g., peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porous, or gelatinous material); andinhalation

In a preferred embodiment, injections or infusions of the siRNA aregiven at or near the site of neovascularization.

The siRNA of the invention can be administered in a single dose or inmultiple doses. Where the administration of the siRNA of the inventionis by infusion, the infusion can be a single sustained dose or can bedelivered by multiple infusions. Injection of the agent directly intothe tissue is at or near the site of neovascularization preferred.Multiple injections of the agent into the tissue at or near the site ofneovascularization are particularly preferred.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA of the invention to a given subject.For example, the siRNA can be administered to the subject once, such asby a single injection or deposition at or near the neovascularizationsite. Alternatively, the siRNA can be administered to a subject once ortwice daily to a subject once weekly for a period of from about three toabout twenty-eight days, more preferably from about seven to about tenweeks. In a preferred dosage regimen, the siRNA is injected at or nearthe site of neovascularization once a day for seven days.

Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of siRNA administered to thesubject can comprise the total amount of siRNA administered over theentire dosage regimen.

The siRNA of the invention are preferably formulated as pharmaceuticalcompositions prior to administering to a subject, according totechniques known in the art. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.As used herein, “pharmaceutical formulations” include formulations forhuman and veterinary use. Methods for preparing pharmaceuticalcompositions of the invention are within the skill in the art, forexample as described in Remington's Pharmaceutical Science, 17th ed.,Mack Publishing Company, Easton, Pa. (1985), the entire disclosure ofwhich is herein incorporated by reference.

The present pharmaceutical formulations comprise an siRNA of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxidants, osmolalityadjusting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example, calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For solid compositions, conventional nontoxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more siRNA of the invention. Apharmaceutical composition for aerosol (inhalational) administration cancomprise 0.01-20% by weight, preferably 1%-10% by weight, of one or moresiRNA of the invention encapsulated in a liposome as described above,and propellant. A carrier can also be included as desired; e.g.,lecithin for intranasal delivery.

The invention will now be illustrated with the following non-limitingexamples. The animal experiments described in Examples 4-6 wereperformed using the University of Pennsylvania institutional guidelinesfor the care and use of animals in research.

Example 1 siRNA Transfection and Hypoxia Induction In Vitro

siRNA Design—

A 19 nt sequence located 329 nt from the 5′ end of human VEGF mRNA waschosen as a target sequence: AAACCTCACCAAGGCCAGCAC (SEQ ID NO: 51). Toensure that it was not contained in the mRNA from any other genes, thistarget sequence was entered into the BLAST search engine provided byNCBI. The use of the BLAST algorithm is described in Altschul et al.(1990), J. Mol. Biol. 215: 403-410 and Altschul et al. (1997), NucleicAcids Res. 25: 3389-3402, the disclosures of which are hereinincorporated by reference in their entirety. As no other mRNA was foundwhich contained the target sequence, an siRNA duplex was synthesized totarget this sequence (Dharmacon Research, Inc., Lafayette, Colo.).

The siRNA duplex had the following sense and antisense strands.

sense: (SEQ ID NO: 77) 5′-accucaccaaggccagcacTT-3′. antisense:(SEQ ID NO: 78) 5′-gugcuggccuuggugagguTT-3′.

Together, the siRNA sense and antisense strands formed a 19 ntdouble-stranded siRNA with TT 3′ overhangs (shown in bold) on eachstrand. This siRNA was termed “Candidate 5” or “Cand5.” Other siRNAwhich target human VEGF mRNA were designed and tested as described forCand5.

An siRNA targeting the following sequence in green fluorescent protein(GFP) mRNA was used as a nonspecific control: GGCTACGTCCAGCGCACC (SEQ IDNO: 79). The siRNA was purchased from Dharmacon (Lafayette, Colo.).

siRNA Transfection and Hypoxia Induction In Vitro—

Human cell lines (293; Hela and ARPE19) were separately seeded into24-well plates in 250 microliters of complete DMEM medium one day priorto transfection, so that the cells were ˜50% confluent at the time oftransfection. Cells were transfected with 2.5 nM Cand5 siRNA, and witheither no siRNA or 2.5 nM non-specific siRNA (targeting GFP) ascontrols. Transfections were performed in all cell lines with the“Transit TKO Transfection” reagent, as recommended by the manufacturer(Mirus).

Twenty four hours after transfection, hypoxia was induced in the cellsby the addition of desferoxamide mesylate to a final concentration of130 micromolar in each well. Twenty four hours post-transfection, thecell culture medium was removed from all wells, and a human VEGF ELISA(R&D systems, Minneapolis, Minn.) was performed on the culture medium asdescribed in the Quantikine human VEGF ELISA protocol available from themanufacturer, the entire disclosure of which is herein incorporated byreference.

As can be seen in FIG. 1, RNAi degradation induced by Cand5 siRNAsignificantly reduces the concentration of VEGF produced by the hypoxic293 and HeLa cells. There was essentially no difference in the amount ofVEGF produced by hypoxic cells treated with either no siRNA or thenon-specific siRNA control. Similar results were also seen with humanARPE19 cells treated under the same conditions. Thus, RNA interferencewith VEGF-targeted siRNA disrupts the pathogenic up-regulation of VEGFin human cultured cells in vitro.

The experiment outlined above was repeated on mouse NIH 3T3 cells usinga mouse-specific VEGF siRNA (see Example 6 below), and VEGF productionwas quantified with a mouse VEGF ELISA (R&D systems, Minneapolis, Minn.)as described in the Quantikine mouse VEGF ELISA protocol available fromthe manufacturer, the entire disclosure of which is herein incorporatedby reference. Results similar to those reported in FIG. 1 for the humancell lines were obtained.

Example 2 Effect of Increasing siRNA Concentration on VEGF Production inHuman Cultured Cells

The experiment outlined in Example 1 was repeated with human 293, HeLaand ARPE19 cells using a range of siRNA concentrations from 10 nM to 50nM. The ability of the Cand5 siRNA to down-regulate VEGF productionincreased moderately up to approximately 13 nM siRNA, but a plateaueffect was seen above this concentration. These results highlight thecatalytic nature of siRNA-mediated RNAi degradation of mRNA, as theplateau effect appears to reflect VEGF production from the few cells nottransfected with the siRNA. For the majority of cells which had beentransfected with the siRNA, the increased VEGF mRNA production inducedby the hypoxia is outstripped by the siRNA-induced degradation of thetarget mRNA at siRNA concentrations greater than about 13 nM.

Example 3 Specificity of siRNA Targeting

NIH 3T3 mouse fibroblasts were grown in 24-well plates under standardconditions, so that the cells were ˜50% confluent one day prior totransfection. The human VEGF siRNA Cand5 was transfected into a NIH 3T3mouse fibroblasts as in Example 1. Hypoxia was then induced in thetransfected cells, and murine VEGF concentrations were measured by ELISAas in Example 1.

The sequence targeted by the human VEGF siRNA Cand5 differs from themurine VEGF mRNA by one nucleotide. As can be seen in FIG. 2, the humanVEGF siRNA has no affect on the ability of the mouse cells toup-regulate mouse VEGF after hypoxia. These results show that siRNAinduced RNAi degradation is sequence-specific to within a one nucleotideresolution.

Example 4 In Vivo Delivery of siRNA to Murine Retinal Pigment EpithelialCells

VEGF is upregulated in the retinal pigment epithelial (RPE) cells ofhuman patients with age-related macular degeneration (ARMD). To showthat functional siRNA can be delivered to RPE cells in vivo, GFP wasexpressed in mouse retinas with a recombinant adenovirus, and GFPexpression was silenced with siRNA. The experiment was conducted asfollows.

One eye from each of five adult C57/Black6 mice (Jackson Labs, BarHarbor, Me.) was injected subretinally as described in Bennett et al.(1996), supra., with a mixture containing ˜1×10⁸ particles of adenoviruscontaining eGFP driven by the CMV promoter and 20 picomoles of siRNAtargeting eGFP conjugated with transit TKO reagent (Mirus).

As positive control, the contralateral eyes were injected with a mixturecontaining ˜1×10⁸ particles of adenovirus containing eGFP driven by theCMV promoter and 20 picomoles of siRNA targeting human VEGF conjugatedwith transit TKO reagent (Mirus). Expression of GFP was detected byfundus ophthalmoscopy 48 hours and 60 hours after injection. Animalswere sacrificed at either 48 hours or 60 hours post-injection. The eyeswere enucleated and fixed in 4% paraformaldehyde, and were preparedeither as flat mounts or were processed into 10 micron cryosections forfluorescent microscopy.

No GFP fluorescence was detectable by ophthalmoscopy in the eyes whichreceived the siRNA targeted to GFP mRNA in 4 out of 5 mice, whereas GFPfluorescence was detectable in the contralateral eye which received thenon-specific control siRNA. A representative flat mount analyzed byfluorescence microscopy showed a lack of GFP fluorescence in the eyewhich received GFP siRNA, as compared to an eye that received thenon-specific control siRNA. Cryosections of another retina showed thatthe recombinant adenovirus efficiently targets the RPE cells, and whenthe adenovirus is accompanied by siRNA targeted to GFP mRNA, expressionof the GFP transgene is halted.

While there is some GFP fluorescence detectable by fluorescencemicroscopy in eyes that received siRNA targeted to GFP mRNA, thefluorescence is greatly suppressed as compared to controls that receivednon-specific siRNA. These data demonstrate that functional siRNA can bedelivered in vivo to RPE cells.

Example 5 In Vivo Expression and siRNA-Induced RNAi Degradation of HumanVEGF in Murine Retinas

In order to demonstrate that siRNA targeted to VEGF functioned in vivo,an exogenous human VEGF expression cassette was delivered to mouse RPEcells via an adenovirus by subretinal injection, as in Example 4. Oneeye received Candy siRNA, and the contralateral eye received siRNAtargeted to GFP mRNA. The animals were sacrificed 60 hourspost-injection, and the injected eyes were removed and snap frozen inliquid N₂ following enucleation. The eyes were then homogenized in lysisbuffer, and total protein was measured using a standard Bradford proteinassay (Roche, Germany). The samples were normalized for total proteinprior to assaying for human VEGF by ELISA as described in Example 1.

The expression of VEGF was somewhat variable from animal to animal. Thevariability of VEGF levels correlated well to those observed in the GFPexperiments of Example 4, and can be attributed to some error frominjection to injection, and the differential ability of adenovirus todelivery the target gene in each animal. However, there was asignificant attenuation of VEGF expression in each eye that receivedVEGF siRNA, as compared to the eyes receiving the non-specific controlsiRNA (FIG. 4). These data indicate that the Cand5 siRNA was potent andeffective in silencing human VEGF expression in murine RPE cells invivo.

Example 6 Inhibition of Choroidal Neovascularization in the Mouse CNVModel

There is evidence that choroidal neovascularization in ARMD is due tothe upregulation of VEGF in the RPE cells. This human pathologiccondition can be modeled in the mouse by using a laser to burn a spot onthe retina (“laser photo-coagulation” or “laser induction”). During thehealing process, VEGF is believed to be up-regulated in the RPE cells ofthe burned region, leading to re-vascularization of the choroid. Thismodel is called the mouse choroidal neovascularization (“CNV”) model.

For rescue of the mouse CNV model, a mouse siRNA was designed thatincorporated a one nucleotide change from the human “Cand5” siRNA fromExample 1. The mouse siRNA specifically targeted mouse VEGF mRNA at thesequence AAACCUCACCAAAGCCAGCAC (SEQ ID NO: 80). Other siRNA that targetmouse VEGF were also designed and tested. The GFP siRNA used as anonspecific control in Example 1 was also used as a non-specific controlhere.

Twenty four hours after laser induction, one eye from each of elevenadult C57/Black6 mice (Jackson Labs, Bar Harbor, Me.) was injectedsubretinally with a mixture containing ˜1×10⁸ particles of adenoviruscontaining LacZ driven by the CMV promoter and 20 picomoles of siRNAtargeting mouse VEGF conjugated with transit TKO reagent (Minis), as inExample 4. As a control, contralateral eyes received a mixturecontaining ˜1×10⁸ particles of adenovirus containing LacZ driven by theCMV promoter and 20 picomoles of siRNA targeting GFP conjugated withtransit TKO reagent (Minis).

Fourteen days after the laser treatment, the mice were perfused withfluorescein and the area of neovascularization was measured around theburn spots. Areas of the burn spots in the contra-lateral eye were usedas a control. The site of neovascularization around the burn spots inanimals that received siRNA targeting mouse VEGF was, on average, ¼ thearea of the control areas. These data support the use of VEGF-directedsiRNA (also called “anti-VEGF siRNA”) for therapy of ARMD.

Example 7 Generation of an Adeno-Associated Viral Vector for Expressionof siRNA

A “cis-acting” plasmid for generating a recombinant AAV vector fordelivering an siRNA of the invention was generated by PCR basedsubcloning, essentially as described in Samulski R et al. (1987), supra.The cis-acting plasmid was called “pAAVsiRNA.”

The rep and cap genes of psub201 were replaced with the followingsequences in this order: a 19 nt sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and a 19 nt antisense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. A schematic representation ofpAAVsiRNA is given if FIG. 5.

A recombinant AAV siRNA vector was obtained by transfecting pAAVsiRNAinto human 293 cells previously infected with E1-deleted adenovirus, asdescribed in Fisher K J et al. (1996), supra. The AAV rep and capfunctions were provided by a trans-acting plasmid pAAV/Ad as describedin Samulski R et al. (1989), supra. Production lots of the recombinantAAV siRNA vector were titered according to the number of genomecopies/ml, as described in Fisher K J et al. (1996), supra.

We claim:
 1. A method of reducing expression of vascular endothelialgrowth factor (VEGF) in a subject comprising administering to thesubject a pharmaceutical composition comprising a dsRNA consisting of a19 to 25 nucleotide sense RNA strand and a 19 to 25 nucleotide antisenseRNA strand, wherein the sense and the antisense RNA strands form an RNAduplex, and wherein the antisense RNA strand consists of a nucleotidesequence that hybridizes to SEQ ID NOs: 20 or
 52. 2. The method of claim1, wherein the antisense RNA strand consists of a nucleotide sequencethat hybridizes to SEQ ID NOs: 20 and
 52. 3. The method of claim 1,wherein the sense and antisense RNA strands forming the RNA duplex arecovalently linked by a single-stranded hairpin.
 4. The method of claim1, wherein the dsRNA further comprises non-nucleotide material.
 5. Themethod of claim 1, wherein the sense and antisense RNA strands arestabilized against nuclease degradation.
 6. The method of claim 1,wherein the RNA duplex has a 3′ overhang.
 7. The method of claim 1,wherein the dsRNA comprises a modified analogue.
 8. The method of claim1, wherein the pharmaceutical composition further comprises a deliveryreagent.
 9. The method of claim 1, wherein the delivery reagentencapsulates the dsRNA.
 10. The method of claim 1, wherein the deliveryreagent is selected from the group consisting of lipofectin,lipofectamine, cellfectin, polycations, and liposomes.
 11. The method ofclaim 8, wherein the delivery reagent is a liposome.
 12. The method ofclaim 9, wherein the delivery reagent is a liposome.
 13. A method oftreating diabetic retinopathy or age-related macular degeneration (ARMD)in a subject comprising administering to the subject a pharmaceuticalcomposition comprising a dsRNA consisting of a 19 to 25 nucleotide senseRNA strand and a 19 to 25 nucleotide antisense RNA strand, wherein thesense and the antisense RNA strands form an RNA duplex, and wherein theantisense RNA strand consists of a nucleotide sequence that hybridizesto SEQ ID NOs: 20 or
 52. 14. The method of claim 13, wherein theantisense RNA strand consists of a nucleotide sequence that hybridizesto SEQ ID NOs: 20 and
 52. 15. The method of claim 13, wherein thesubject is in need of such treatment.
 16. The method of claim 13,wherein the method decreases the expression of vascular endothelialgrowth factor (VEGF).
 17. The method of claim 13, wherein the methoddecreases the protein expression VEGF in the subject.
 18. The method ofclaim 13, wherein the method decreases the amount of VEGF mRNA in thesubject.
 19. The method of claim 13, wherein the pharmaceuticalcomposition further comprises a delivery reagent.
 20. The method ofclaim 13, wherein the delivery reagent encapsulates the dsRNA.